Malaria transmission in the field requires the production of sexual stage Plasmodium parasites, called gametocytes, that once taken up by an Anopheles mosquito fertilize and differentiate into infectious sporozoites. Sporozoites collect in the mosquito salivary gland and can be introduced to a new host during a blood meal. As would be expected, higher gametocyte levels in the human host's peripheral blood increase the chance of productive transmission to a mosquito, but gametocyte levels vary between individuals and the factors that determine carriage are unknown. No tools are available to identify individuals that carry immature P. falciparum gametocytes to determine the efficiency of stage V gametocyte development or to target carriers for treatment before they become infectious. In a recent field study of 260 symptomatic malaria patients in Ghana we developed a new assay to quantify circulating gc-rings and found a wide range of gametocyte conversion rates. In 20% of the samples the ratio of gc-rings to asexual rings was > 4.9% with a high of 70%, while 24% of the samples had no gc-rings, which clearly demonstrates variation in gametocytogenesis between patients. Using this data we found that gametocyte conversion was positively associated with parasitemia, while body temperature was negatively associated. We also found high gametocyte conversion was associated with a specific allele of a gene previously reported to be critical for gametocyte production in vitro, gametocyte development 1 protein (gdv1) and the upregulation of three enzymes. We hypothesize that these factors and genes are causally associated with P. falciparum gametocytogenesis and plan to test this directly in vitro (Aims 1 and 2). We will also extend our in vivo analysis of gametocyte dynamics to one of the major infectious reservoirs in West Africa, asymptomatic children 5-15 years old (Aim 3). Children will be followed longitudinally from the start to the end of the malaria season in Ghana (May-Nov) and again in the dry season (Apr) over the course of 2 years in 2 different sites. This prospective longitudinal study design will allow for the first time the evaluation of asexual and gc-ring production as well as gametocyte maturation/survival and host parameters in the same individual over time. The two week sampling period is the same as the time needed for a P. falciparum gc-merozoite invaded RBC to mature to a stage V gametocyte. The direct comparison of gc-rings and stage V gametocytes produced two weeks later will indicate whether gc-ring levels can be used to predict the number of stage V gametocytes found in the circulation two weeks later or whether some individuals effectively clear sequestered gametocytes prior to maturation. Evidence for gametocyte clearance would provide new targets for transmission-blocking interventions. Together, the data from all three aims will allow us to determine whether the host environment or the parasite or host genotype are major drivers of malaria transmission, providing critical information for the design of malaria control strategies.